Cells contain thousands of multimolecular complexes which work together much like miniature factory machines. A detailed understanding of the structure and function of these molecular devices is a problem of great interest in cell biology. Our research efforts focus on electron cryo-microscopic analysis of single particles, a powerful method to determine the three-dimensional architectures of complex cellular assemblies. We have defined and interpreted the structure of an icosahedral pyruvate dehydrogenase multienzyme complex, a prototypical example of a multi-step catalytic machine which couples the activity of three component enzymes (E1, E2, and E3) in the oxidative decarboxylation of pyruvate to generate acetyl CoA at the junction of glycolysis and the tricarboxylic acid cycle. The three-dimensional model for a 11 MDa, icosahedral PDH complex, composed of 60 E2 enzymes and 60 E1 enzymes, was obtained by combining a 28 structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual components of the complex (Milne et al. EMBO J. 21, 5587, 2002). Analysis of the model provides a number of novel insights into the design and function of this molecular machine. A key feature is that the E1 molecules are located on the periphery in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 enzyme to access multiple E1 active sites from inside the icosahedral complex. This unanticipated architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement, which we propose is achieved by the motion of the lipoyl domain in the restricted annular region between the inner and outer cores of the complex. We are currently refining a second PDH complex comprised of 60 E2 enzymes and 60 E3 enzymes to determine the structural basis of why in vivo the inner icosahedron of 60 E2 molecules is occupied with only 48 E1 molecules and 6 E3 molecules to form the outer protein shell. Our three-dimensional reconstruction this complex indicates that, similar to the E1E2 complex described above, an annular gap of 75 exists between the inner core and the outer shell of E3 homodimers. Image analysis of partial occupancy complexes, formed by decorating the E2 core with 10, 20, 40 or 60 E1 tetramers or with 10, 20, 40 or 60 E3 homodimers, also indicates a 75-95 separation of the E2 and E1 or E2 and E3 densities. Thus, interactions occurring between the enzymes of the outer protein shell are not responsible for maintenance of the size of the complex. Rather, the E2 inner linkers that connect the E2 catalytic domains to the E2 peripheral-subunit binding domains must be fairly rigid radial spokes that provide a scaffold for an organization of E1 and E3 molecules. E3 localizes slightly closer to the core, suggesting that the swinging lipoyl domain can effectively access the active sites of all three enzymes, without leaving the annular space. This architecture provides further evidence of the highly efficient mechanism for active site coupling during both the production of acetyl CoA and the subsequent enzyme regeneration step that is required to initiate a new cycle of acetyl CoA production. We are also working actively to identify conditions that lead to outstanding microscopic images, to develop methods to select and accurately align the best molecular images for three-dimensional reconstructions, to reliably interpret these structures, and to develop automated procedures to facilitate the process of obtaining high quality three dimensional models of macromolecular complexes. To this end, we have 1) developed algorithms to collect data automatically on the Tecnai series of electron microscopes, 2) characterized the properties of a 4000 x 4000 pixel digital CCD camera, and assessed the quality of the three-dimensional molecular models constructed from CCD digital images, 3) developed a "core-weighting" method, combined with a grid-threading Monte Carlo approach to enhance the ability to reliably identify the best fit of atomic coordinates of individual components into low resolution maps of larger complexes that are typical of structures determined with the use of single particle electron microscopy and 4) optimized methods for the computational analysis of molecular images . We have demonstrated that the combination of iterative refinement and stringent molecular sorting can be an effective method to obtain substantial improvements in map quality of the 1.8 MDa icosahedral E2 catalytic core. From a starting set of 42,945 images of the core complex, we have shown that using only the best 139 particles in the data set produces a map that is superior to those constructed with greater numbers of images, and that many of the alpha-helices in the structure can be unambiguously visualized in a map constructed from as few as 9 particles. Thus, stringent computational sorting of molecular images may help overcome the significant limitations posed by heterogeneity in image quality and in protein conformation that are inherent to large data sets of images. Application of this method and improved fitting algorithms to facilitate accurate docking of X-ray coordinates of individual component proteins into the density maps obtained by cryo electron microscopy should enhance visualization of important macromolecular complexes present in normal and malignant cells.